Of late, Analytical Quality by Design (AQbD) has been gaining increased acceptance in the industrial, academic and regulatory circles. Considered as a science and risk-based approach, AQbD provides rational understanding of the critical method parameters (CMPs) affecting the critical analytical attributes (CAAs) of an analytical method. The present work aims at systematic development of a simple, rapid and highly sensitive bioanalytical high-performance thin-layer densitometry method for the analysis of mangiferin. Initially, the quality target method profile (QTMP) was defined and critical analytical attributes (CAAs) were earmarked. Preliminary studies were conducted for selecting the suitable mobile phase mixture, followed by primary and secondary screening studies employing D-optimal design and Plackett-Burman design, respectively for selecting the ideal mobile phase composition and prioritizing the critically influential method parameters on the CAAs. The CAAs chosen included retardation factor (Rf), peak height, capacity factor, theoretical plates and separation number. Response Surface Methodology (RSM) was conducted as per the face centered cubic design (FCCD) for optimizing volume loaded and plate dimension as the critical method parameters (CMPs) selected initially from the screening studies. The mobile phase containing mixture of ethyl acetate: acetic acid: formic acid: water in 7:1:1:1, v/v/v/v ratio was finally selected as the optimized combination owing to apt chromatographic separation for mangiferin at 262 nm with Rf 0.68±0.02, with all other parameters being within the acceptance limits. Method validation studies revealed high linearity for mangiferin in the concentration range of 50–800 ng/band with r2 =0.998 ± 0.005. The developed method showed high accuracy, precision, ruggedness, robustness, specificity, sensitivity, selectivity, recovery, detection limit (12.1 ng/band) and quantification limit (36.6 ng/band). The bioanalytical method for analysis of mangiferin in plasma revealed the presence of well-resolved peaks and high recovery of mangiferin.